The immediate goal of this project is to investigate difference in DNA methylation between fusion-positive and fusion-negative rhabdomyosarcoma (RMS) tumors. During FY 2013, a staff scientist commenced analysis of this DNA methylation project until that individual left the laboratory in November 2012. A research geneticist in the laboratory then continued these studies from March 2013 to the present. Various bioinformatic strategies were applied to the existing Infinium Human Methylation 27 BeadChip data to identify genes that are differentially methylated between fusion-positive and fusion-negative RMS tumors. Supervised analysis was used to compare mean methylation levels for each gene in the fusion-negative and fusion-positive tumors, and then to determine those genes whose mean methylation levels were significantly different between the two groups of tumors. Genes were found for which there was both evidence of hypermethylation in fusion-positive RMS and hypomethylation in fusion-negative RMS, and other genes were found for which there was evidence of hypomethylation in fusion-positive RMS and hypermethylation in fusion-negative RMS. As each methylation measurement applies to a single CpG site, confidence and interest increased when the relationship was found for more than one CpG in the same gene. Our next goal was to validate the differential methylation of selected genes by determining the methylation status of the larger CpG-containing regulatory region in fusion-negative and positive RMS tumors. We selected pyrosequencing because of its ability to quantitatively assess the extent of methylation of multiple CpG sites within a selected CpG-containing region. In particular, this technology measures the C:T ratio at multiple cytosines within CpG dinucleotides in a bisulfite-treated DNA sample and thereby determines the methylation status of these sites within the CpG-containing region. As this procedure requires amplification of the selected CpG-rich regions with flanking primers that do not contain CpG dinucleotides (and thus do not have a variable sequence dependent on methylation status), we used existing PyroMark CpG Assays (Qiagen) when available or used the PyroMark Assay Design Software 2.0 software to design custom primers. Based on these various requirements, we selected four genes that are potentially hypermethylated in fusion-positive RMS tumors and four genes that are potentially hypermethylated in fusion-negative tumors. We initially used RMS tumor cell lines to work out this methodology. Our cell line results indicate that the methylation differences between fusion-negative and fusion-positive RMS tumors, as determined on the Infinium Human Methylation 27 BeadChip, are not fully maintained in RMS cell lines. We will next use DNA from RMS tumor samples, and in particular, we will start with DNA from the samples assayed on the 27 BeadChip. In a separate set of studies, we assayed expression of four genes for which there was at least one CpG site that showed a substantial difference in methylation between fusion-positive and fusion-negative RMS tumors. For one gene in which one of two CpG sites showed substantially higher methylation in fusion-positive than fusion negative tumors, the expression was significantly higher in the fusion-negative tumors. For two genes in which multiple CpG sites demonstrated higher methylation in fusion-positive than fusion-negative tumors, the expression was surprisingly higher in the fusion-positive tumors. Finally, for one gene in which one of two CpG sites showed substantially higher methylation in fusion-negative than fusion-positive tumors, there was no significant difference in expression between the two fusion subsets.